Targeting Transcriptional and Translational Hindrances in a Modular T7RNAP Expression System in Engineered Pseudomonas putida.

The T7 RNA polymerase is considered one of the most popular tools for heterologous gene expression in the gold standard biotechnological host Escherichia coli. However, the exploitation of this tool in other prospective hosts, such as the biotechnologically relevant bacterium Pseudomonas putida, is still very scarce. The majority of the existing T7-based systems in P. putida show low expression strengths and possess only weak controllability. A fundamental understanding of these systems is necessary in order to design robust and predictable biotechnological processes. To fill this gap, we established and characterized a modular T7 RNA polymerase-based system for heterologous protein production in P. putida, using the enhanced Green Fluorescent Protein (eGFP) as an easy-to-quantify reporter protein. We have effectively targeted the limitations associated with the initial genetic setup of the system, such as slow growth and low protein production rates. By replacing the T7 phage-inherent TΦ terminator downstream of the heterologous gene with the synthetic tZ terminator, growth and protein production rates improved drastically, and the T7 RNA polymerase system reached a productivity level comparable to that of an intrinsic RNA polymerase-based system. Furthermore, we were able to show that the system was saturated with T7 RNA polymerase by applying a T7 RNA polymerase ribosome binding site library to tune heterologous protein production. This saturation indicates an essential role for the ribosome binding sites of the T7 RNA polymerase since, in an oversaturated system, cellular resources are lost to the synthesis of unnecessary T7 RNA polymerase. Eventually, we combined the experimental data into a model that can predict the eGFP production rate with respect to the relative strength of the ribosome binding sites upstream of the T7 gene.

Immobilization of PETase enzymes on magnetic iron oxide nanoparticles for the decomposition of microplastic PET.

Polyethylene terephthalate (PET) is responsible for a large amount of environmental contamination with microplastics. Based on its high affinity, the PET degrading enzyme PETase can be immobilized on superparamagnetic iron oxide nanoparticles through a His-tag. The His-tag increases enzyme stability, and allows magnetic separation for recovery. Multiple recycling steps are possible and microplastic particles can be decomposed depending on the PET's crystallinity. The separation or decomposition of PET allows for a sustainable way to remove microplastic from water.

In-Depth Computational Analysis of Natural and Artificial Carbon Fixation Pathways.

In the recent years, engineering new-to-nature CO2- and C1-fixing metabolic pathways made a leap forward. New, artificial pathways promise higher yields and activity than natural ones like the Calvin-Benson-Bassham (CBB) cycle. The question remains how to best predict their in vivo performance and what actually makes one pathway "better" than another. In this context, we explore aerobic carbon fixation pathways by a computational approach and compare them based on their specific activity and yield on methanol, formate, and CO2/H2 considering the kinetics and thermodynamics of the reactions. Besides pathways found in nature or implemented in the laboratory, this included two completely new cycles with favorable features: the reductive citramalyl-CoA cycle and the 2-hydroxyglutarate-reverse tricarboxylic acid cycle. A comprehensive kinetic data set was collected for all enzymes of all pathways, and missing kinetic data were sampled with the Parameter Balancing algorithm. Kinetic and thermodynamic data were fed to the Enzyme Cost Minimization algorithm to check for respective inconsistencies and calculate pathway-specific activities. The specific activities of the reductive glycine pathway, the CETCH cycle, and the new reductive citramalyl-CoA cycle were predicted to match the best natural cycles with superior product-substrate yield. However, the CBB cycle performed better in terms of activity compared to the alternative pathways than previously thought. We make an argument that stoichiometric yield is likely not the most important design criterion of the CBB cycle. Still, alternative carbon fixation pathways were paretooptimal for specific activity and product-substrate yield in simulations with C1 substrates and CO2/H2 and therefore hold great potential for future applications in Industrial Biotechnology and Synthetic Biology.

Corrigendum to "In-Depth Computational Analysis of Natural and Artificial Carbon Fixation Pathways".

[This corrects the article DOI: 10.34133/2021/9898316.].

Trehalose production by Cupriavidus necator from CO2 and hydrogen gas.

In this work, the hydrogen-oxidizing bacterium Cupriavidus necator H16 was engineered for trehalose production from gaseous substrates. First, it could be shown that C. necator is a natural producer of trehalose when stressed with sodium chloride. Bioinformatic investigations revealed a so far unknown mode of trehalose and glycogen metabolism in this organism. Next, it was found that expression of the sugar efflux transporter A (setA) from Escherichia coli lead to a trehalose leaky phenotype of C. necator. Finally, the strain was characterized under autotrophic conditions using a H2/CO2/O2-mixture and other substrates reaching titers of up to 0.47 g L-1 and yields of around 0.1 g g-1. Taken together, this process represents a new way to produce sugars with high areal efficiency. With further metabolic engineering, an application of this technology for the renewable production of trehalose and other sugars, as well as for the synthesis of 13C-labeled sugars seems promising.

A Nitrate-Blind P. putida Strain Boosts PHA Production in a Synthetic Mixed Culture.

One of the major challenges for the present and future generations is to find suitable substitutes for the fossil resources we rely on today. In this context, cyanobacterial carbohydrates have been discussed as an emerging renewable feedstock in industrial biotechnology for the production of fuels and chemicals. Based on this, we recently presented a synthetic bacterial co-culture for the production of medium-chain-length polyhydroxyalkanoates (PHAs) from CO2. This co-cultivation system is composed of two partner strains: Synechococcus elongatus cscB which fixes CO2, converts it to sucrose and exports it into the culture supernatant, and a Pseudomonas putida strain that metabolizes this sugar and accumulates PHAs in the cytoplasm. However, these biopolymers are preferably accumulated under conditions of nitrogen limitation, a situation difficult to achieve in a co-culture as the other partner, at best, should not perceive any limitation. In this article, we will present an approach to overcome this dilemma by uncoupling the PHA production from the presence of nitrate in the medium. This is achieved by the construction of a P. putida strain that is no longer able to grow with nitrate as nitrogen source -is thus nitrate blind, and able to grow with sucrose as carbon source. The deletion of the nasT gene encoding the response regulator of the NasS/NasT two-component system resulted in such a strain that has lost the ability use nitrate, but growth with ammonium was not affected. Subsequently, the nasT deletion was implemented in P. putida cscRABY, an efficient sucrose consuming strain. This genetic engineering approach introduced an artificial unilateral nitrogen limitation in the co-cultivation process, and the amount of PHA produced from light and CO2 was 8.8 fold increased to 14.8% of its CDW compared to the nitrate consuming reference strain. This nitrate blind strain, P. putidaΔnasT attTn7:cscRABY, is not only a valuable partner in the co-cultivation but additionally enables the use of other nitrate containing substrates for medium-chain-length PHA production, like for example waste-water.

Engineering sucrose metabolism in Pseudomonas putida highlights the importance of porins.

Using agricultural wastes as a substrate for biotechnological processes is of great interest in industrial biotechnology. A prerequisite for using these wastes is the ability of the industrially relevant microorganisms to metabolize the sugars present therein. Therefore, many metabolic engineering approaches are directed towards widening the substrate spectrum of the workhorses of industrial biotechnology like Escherichia coli, yeast or Pseudomonas putida. For instance, neither xylose or arabinose from cellulosic residues, nor sucrose, the main sugar in waste molasses, can be metabolized by most E. coli and P. putida wild types. We evaluated a new, so far uncharacterized gene cluster for sucrose metabolism from Pseudomonas protegens Pf-5 and showed that it enables P. putida to grow on sucrose as the sole carbon and energy source. Even when integrated into the genome of P. putida, the resulting strain grew on sucrose at rates similar to the rate of the wild type on glucose - making it the fastest growing, plasmid-free P. putida strain known so far using sucrose as substrate. Next, we elucidated the role of the porin, an orthologue of the sucrose porin ScrY, in the gene cluster and found that in P. putida, a porin is needed for sucrose transport across the outer membrane. Consequently, native porins were not sufficient to allow unlimited growth on sucrose. Therefore, we concluded that the outer membrane can be a considerable barrier for substrate transport, depending on strain, genotype and culture conditions, all of which should be taken into account in metabolic engineering approaches. We additionally showed the potential of the engineered P. putida strains by growing them on molasses with efficiencies twice as high as obtained with the wild-type P. putida. This can be seen as a further step towards the production of low-value chemicals and biofuels with P. putida from alternative and more affordable substrates in the future.

Continuous conversion of CO2/H2 with Clostridium aceticum in biofilm reactors.

A lab-scale stirred-tank bioreactor was reversibly retrofitted to a packed-bed and a trickle-bed biofilm reactor to study and compare the conversion of CO2/H2 with immobilised Clostridiumaceticum. The biofilm reactors were characterised and their functionality confirmed. Up to 8.6 g of C. aceticum were immobilised onto 300 g sintered ceramic carrier material, proving biofilm formation to be a robust means for cell retention of C. aceticum. Continuous CO2/H2-fermentation studies were performed with both biofilm reactor configurations as function of dilution rates, partial gas pressures and gas flow rates. The experiments showed that in the packed-bed biofilm reactor, the acetate space-time yield was independent of the dilution rate, because of low H2 gas-liquid mass transfer rates (≤17 mmol H2 L-1 h-1). The continuous operation of the trickle-bed biofilm reactor increased the gas-liquid mass transfer rates to up to 56 mmol H2 L-1 h-1. Consequently, the acetate space-time yield of up to 14 mmol acetate L-1 h-1 was improved 3-fold at hydrogen conversions of up to 96%.

An automated and parallelised DIY-dosing unit for individual and complex feeding profiles: Construction, validation and applications.

Since biotechnological research becomes more and more important for industrial applications, there is an increasing need for scalable and controllable laboratory procedures. A widely used approach in biotechnological research to improve the performance of a process is to vary the growth rates in order to find the right balance between growth and the production. This can be achieved by the application of a suitable feeding strategy. During this initial bioprocess development, it is beneficial to have at hand cheap and easy setups that work in parallel (e.g. in shaking flasks). Unfortunately, there is a gap between these easy setups and defined and controllable processes, which are necessary for up-scaling to an industrial relevant volume. One prerequisite to test and evaluate different process strategies apart from batch-mode is the availability of pump systems that allow for defined feeding profiles in shaking flasks. To our knowledge, there is no suitable dosing device on the market which fulfils the requirements of being cheap, precise, programmable, and parallelizable. Commercially available dosing units are either already integrated in bioreactors and therefore inflexible, or not programmable, or expensive, or a combination of those. Here, we present a LEGO-MINDSTORMS-based syringe pump, which has the potential of being widely used in daily laboratory routine due to its low price, programmability, and parallelisability. The acquisition costs do not exceed 350 € for up to four dosing units, that are independently controllable with one EV3 block. The system covers flow rates ranging from 0.7 µL min-1 up to 210 mL min-1 with a reliable flux. One dosing unit can convey at maximum a volume of 20 mL (using all 4 units even up to 80 mL in total) over the whole process time. The design of the dosing unit enables the user to perform experiments with up to four different growth rates in parallel (each measured in triplicates) per EV3-block used. We estimate, that the LEGO-MINDSTORMS-based dosing unit with 12 syringes in parallel is reducing the costs up to 50-fold compared to a trivial version of a commercial pump system (~1500 €) which fits the same requirements. Using the pump, we set the growth rates of a E. coli HMS174/DE3 culture to values between 0.1 and 0.4 h-1 with a standard deviation of at best 0.35% and an average discrepancy of 13.2%. Additionally, we determined the energy demand of a culture for the maintenance of the pTRA-51hd plasmid by quantifying the changes in biomass yield with different growth rates set. Around 25% of total substrate taken up is used for plasmid maintenance. To present possible applications and show the flexibility of the system, we applied a constant feed to perform microencapsulation of Pseudomonas putida and an individual dosing profile for the purification of a his-tagged eGFP via IMAC. This smart and versatile dosing unit, which is ready-to-use without any prior knowledge in electronics and control, is affordable for everyone and due to its flexibility and broad application range a valuable addition to the laboratory routine.

pTRA - A reporter system for monitoring the intracellular dynamics of gene expression.

The presence of standardised tools and methods to measure and represent accurately biological parts and functions is a prerequisite for successful metabolic engineering and crucial to understand and predict the behaviour of synthetic genetic circuits. Many synthetic gene networks are based on transcriptional circuits, thus information on transcriptional and translational activity is important for understanding and fine-tuning the synthetic function. To this end, we have developed a toolkit to analyse systematically the transcriptional and translational activity of a specific synthetic part in vivo. It is based on the plasmid pTRA and allows the assignment of specific transcriptional and translational outputs to the gene(s) of interest (GOI) and to compare different genetic setups. By this, the optimal combination of transcriptional strength and translational activity can be identified. The design is tested in a case study using the gene encoding the fluorescent mCherry protein as GOI. We show the intracellular dynamics of mRNA and protein formation and discuss the potential and shortcomings of the pTRA plasmid.

Photoautotrophic production of polyhydroxyalkanoates in a synthetic mixed culture of Synechococcus elongatus cscB and Pseudomonas putida cscAB.

BACKGROUND: One of the major challenges for the present and future generations is to find suitable substitutes for the fossil resources we rely on today. Cyanobacterial carbohydrates have been discussed as an emerging renewable feedstock in industrial biotechnology for the production of fuels and chemicals, showing promising production rates when compared to crop-based feedstock. However, intrinsic capacities of cyanobacteria to produce biotechnological compounds are limited and yields are low. RESULTS: Here, we present an approach to circumvent these problems by employing a synthetic bacterial co-culture for the carbon-neutral production of polyhydroxyalkanoates (PHAs) from CO2. The co-culture consists of two bio-modules: Bio-module I, in which the cyanobacterial strain Synechococcus elongatus cscB fixes CO2, converts it to sucrose, and exports it into the culture supernatant; and bio-module II, where this sugar serves as C-source for Pseudomonas putida cscAB and is converted to PHAs that are accumulated in the cytoplasm. By applying a nitrogen-limited process, we achieved a maximal PHA production rate of 23.8 mg/(L day) and a maximal titer of 156 mg/L. We will discuss the present shortcomings of the process and show the potential for future improvement. CONCLUSIONS: These results demonstrate the feasibility of mixed cultures of S. elongatus cscB and P. putida cscAB for PHA production, making room for the cornucopia of possible products that are described for P. putida. The construction of more efficient sucrose-utilizing P. putida phenotypes and the optimization of process conditions will increase yields and productivities and eventually close the gap in the contemporary process. In the long term, the co-culture may serve as a platform process, in which P. putida is used as a chassis for the implementation of synthetic metabolic pathways for biotechnological production of value-added products.

Metabolic engineering to expand the substrate spectrum of Pseudomonas putida toward sucrose.

Sucrose is an important disaccharide used as a substrate in many industrial applications. It is a major component of molasses, a cheap by-product of the sugar industry. Unfortunately, not all industrially relevant organisms, among them Pseudomonas putida, are capable of metabolizing sucrose. We chose a metabolic engineering approach to circumvent this blockage and equip P. putida with the activities necessary to consume sucrose. Therefore, we constructed a pair of broad-host range mini-transposons (pSST - sucrose splitting transposon), carrying either cscA, encoding an invertase able to split sucrose into glucose and fructose, or additionally cscB, encoding a sucrose permease. Introduction of cscA was sufficient to convey sucrose consumption and the additional presence of cscB had no further effect, though the sucrose permease was built and localized to the membrane. Sucrose was split extracellularly by the activity of the invertase CscA leaking out of the cell. The transposons were also used to confer sucrose consumption to Cupriavidus necator. Interestingly, in this strain, CscB acted as a glucose transporter, such that C. necator also gained the ability to grow on glucose. Thus, the pSST transposons are functional tools to extend the substrate spectrum of Gram-negative bacterial strains toward sucrose.

Model-supported phototrophic growth studies with Scenedesmus obtusiusculus in a flat-plate photobioreactor.

Light-dependent growth of microalgae can vary remarkably depending on the cultivation system and microalgal strain. Cell size and the pigmentation of each strain, as well as reactor geometry have a great impact on absorption and scattering behavior within a photobioreactor. In this study, the light-dependent, cell-specific growth kinetics of a novel green algae isolate, Scenedesmus obtusiusculus, was studied in a LED-illuminated flat-plate photobioreactor on a lab-scale (1.8 L, 0.09 m2 ). First, pH-controlled batch processes were performed with S. obtusiusculus at different constant incident photon flux densities. The best performance was achieved by illuminating S. obtusiusculus with 1400 µmol photons m-2 s-1 at the surface of the flat-plate photobioreactor, resulting in the highest biomass concentration (4.95 ± 0.16 gCDW L-1 within 3.5 d) and the highest specific growth rate (0.22 h-1 ). The experimental data were used to identify the kinetic parameters of different growth models considering light inhibition for S. obtusiusculus. Light attenuation within the flat-plate photobioreactor was considered by varying light transfer models. Based on the identified kinetic growth model of S. obtusiusculus, an optimum growth rate of 0.22 h-1 was estimated at a mean integral photon flux density of 1072 µmol photons m-2 s-1 with the Beer-Lambert law and 1590 µmol photons m-2 s-1 with Schuster's light transfer model in the flat-plate photobioreactor. LED illumination was, thus, increased to keep the identified optimum mean integral photon flux density constant in the batch process assuming Schuster's light transfer model. Compared to the same constant incident photon flux density (1590 µmol photons m-2 s-1 ), biomass concentration was up to 24% higher using the lighting profile until a dry cell mass concentration of 14.4 ± 1.4 gCDW L-1 was reached. Afterward, the biomass concentration remained constant, whereas cell growth continued in the batch process with constant incident photon flux density. Finally, biomass concentration was 15.5 ± 1.5 gCDW L-1 and, thus, 7% higher compared to the corresponding batch process with lighting profile. Biotechnol. Bioeng. 2017;114: 308-320. © 2016 Wiley Periodicals, Inc.

Time Hierarchies and Model Reduction in Canonical Non-linear Models.

The time-scale hierarchies of a very general class of models in differential equations is analyzed. Classical methods for model reduction and time-scale analysis have been adapted to this formalism and a complementary method is proposed. A unified theoretical treatment shows how the structure of the system can be much better understood by inspection of two sets of singular values: one related to the stoichiometric structure of the system and another to its kinetics. The methods are exemplified first through a toy model, then a large synthetic network and finally with numeric simulations of three classical benchmark models of real biological systems.